The present invention concerns a method for determining the degranulation activity of basophilic granulocytes in a sample as well as a reagent kit that is suitable for this. In addition the invention also concerns a method for diagnosing allergic hypersensitivity and the response to a hyposensitization.
The classical classification of allergies according to Coombs and Gell still largely applies nowadays as a basis for understanding the allergic type 1 reaction of the immediate type and is clearly defined by an IgE-mediated immune response. T cells with a particular cytokine pattern (interleukin 4 and interleukin 5) play a special role in this. As a result B lymphocytes are stimulated to switch to IgE production which only occurs at very low levels in the serum and only develops its fatal effect after cell binding to high affinity receptors and subsequent cell degranulation. The cytokines, in particular interleukin 5, additionally cause an increased maturation of promyelocytes into eosinophilic granulocytes which cause delayed damage especially to the bronchial system by the release of toxic proteins from their granula. The physiological relevance of the type 1 allergy is still completely unclear as well as the reason why the T cells secrete a cytokine pattern for an IgE response.
The determination of specific IgE antibodies in the serum (RAST, CAP etc.) is well established and is especially helpful when known allergens need to be tested or when a skin test is dangerous or technically impossible for example in skin diseases or under the influence of drugs. In these methods the allergens are bound covalently or with high affinity to matrices with a large surface (sponges, beads, paper discs), subsequently incubated with patient serum and then, after washing, a detection is carried out using a radioactive or enzyme-labelled anti-human-IgE antibody. The amount of bound IgE is stated semiquantitatively in so-called RAST and CAP classes or quantitatively in kU IgE/l. The evaluation can be carried out radiometrically (RAST) or for example, fluorimetrically (CAP, enzyme with a fluorogenic substrate).
The advantages of these tests are that they are easy to carry out and there is a good correlation with the gold standard skin test for aeroallergens and so-called atopens. However, serious disadvantages must be accepted. These disadvantages are for example the lack of rare allergens and often epitope changes due to coupling of the allergens to the matrix. In addition a carrier molecule such as human serum albumin is required for smaller allergens (haptens). Further difficulties are caused by the fact that the specificities of serum IgE and membrane-bound IgE on basophils and mast cells are not necessarily the same since only a slow exchange takes place. Also the threshold of degranulation readiness (releasability) is not detected. Correlation with the response to a hyposensitization in the form of an IgE decrease is only fairly satisfactory for insect venom allergens i.e. in the majority of cases there is no explanation. Furthermore the specific IgE values must be put into perspective in the case of atopic patients with a high IgE level i.e. the total IgE level of the patient must be known. The severity of the clinical picture hardly correlates with the level of the specific IgE concentration.
Ready-made test strips have been developed as a simplified version of this method on which the various allergens are applied. They are evaluated by comparing the blue colour that forms with a colour scale. Inhalation antigens which produce a reaction in the higher RAST classes are readily detected. Otherwise there is little comparative data. The test is primarily conceived for allergists in private practice who do not have a large laboratory.
Due to the problems mentioned above in determining specific serum IgE, tests have been developed in which the basophilic granulocytes are degranulated after stimulation with an allergen and subsequently the released components of the granula are determined in the supernatant. Such components are for example histamine (Immunotech Co. IBL), leukotrienes (CAST-ELISA, Biermann Co.) or tryptase (Pharmacia Co.). These assays are designed as enzyme immunoassays in a batch procedure and require a duration of two days due to overnight incubation steps. A further disadvantage with regard to the histamine determination is the acylation step for the released histamine as well as a frequently excessively high histamine content of the allergen solutions due to bacterial contaminations. Furthermore for economic reasons it is necessary to collect the supernatants of several patients which delays the assessment. The degranulation must, however, be carried out on fresh whole blood on the day of blood withdrawal.
Therefore attempts have already been made to detect the degranulation cytometrically. In one approach the absorption of basophilic granula was determined with a Technicon H6000 measuring instrument. In this case the measurement points were counted by hand on the screen (Nilsson, Eur. J. Haematol, 45 suppl 53, 50-54, 1990). Alternatively the axial loss of light is measured after staining with toluidine blue in an Ortho cytofluorograph (Nakagawa et al. (Allergy 36, 39-47, 1981). A further approach is to carry out a flow-cytometric two colour immunofluorescence method in which differently labelled anti-CD45 and anti-IgE antibodies are used (Gane et al., Cytometry 19, 361-365, 1995). This method is based on a relative increase of the expression density of CD45 and a reduction of the IgE antibody expression in degranulation. However, a disadvantage of this method is that the change in the CD45 expression density is only small.
None of these cytometric methods has been introduced for routine use due to the said disadvantages since they are tied to special, not generally available instruments or the signal differences are very slight. Furthermore it is difficult to measure the basophils since they are rare in the blood and are very difficult to distinguish from other leucocytes on the basis of scattered light properties alone.
Hence the object that forms the basis of the present invention is to provide a new method for determining the degranulation activity of basophilic granulocytes which can be carried out simply and rapidly and can be performed with conventional flow cytometers which are widely used in hospitals and laboratory practices.